DTIC ADA507006: Developmental and Environmental Regulation of AaeIAP1 Transcript in Aedes aegypti

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MOLECULARBIOLOGY/GENOMICS
Developmental and Environmental Regulation of AaeIAP1 Transcript
in Aedes aegypti
JULIAW.PRIDGEON,1LIMINGZHAO,JAMESJ.BECNEL,GARYG.CLARK,
ANDKENNETHJ.LINTHICUM
CenterforMedical,Agricultural,andVeterinaryEntomology,USDAÐARS,1600SW23rdDrive,Gainesville,FL32608
J.Med.Entomol.45(6):1071Ð1079(2008)
ABSTRACT Apoptosis(programmedcelldeath)isatightlyregulatedphysiologicalprocess.The
inhibitors of apoptosis proteins (IAPs) are key regulators for apoptosis. An inhibitor of apoptosis
protein gene IAP1 was recently cloned fromAedes aegypti (L.) (AaeIAP1, GenBank accession no.
DQ993355); however, it is not clear whether AaeIAP1 is developmentally and environmentally
regulated.Inthisstudy,weappliedquantitativepolymerasechainreaction(PCR)toinvestigatethe
expression levels of the AaeIAP1 transcript in different developmental stages and under different
environmental conditions. Our results revealed that the expression of the AaeIAP1 transcript was
detectableinalllifestagesofAe.aegypti,withsigniÞcantlyhigherlevelsinpupalandadultstagesthan
inlarvalstages.Furthermore,whenAe.aegyptiwasexposedtoallstressfulenvironmentalconditions
(e.g.,lowandhightemperatures,UVradiation,acetone,andpermethrininsecticidetreatment),the
expressionlevelofAaeIAP1transcriptwasincreasedsigniÞcantly.OurresultssuggestthatAaeIAP1
mightplayanimportantroleinboththephysiologicaldevelopmentofAe.aegyptiandstress-induced
apoptosis.
KEYWORDS inhibitorofapoptosis,inhibitorofapoptosisprotein,Aedesaegypti,quantitativePCR,
developmentalregulation
Apoptosisisanevolutionarilyconservedeventinthe Ininsects,Drosophilaistheleadinginsectmodelfor
development of multicellular organisms to remove thestudyofapoptosisregulation.Todate,fourDro-
unwanted,damaged,mutated,orinfectedcells(Liu sophila IAPs (DIAP1, DIAP2, Deterin, and Bruce)
andHengartner1999,TwomeyandMcCarthy2005). havebeenreported(Hayetal.1995,Jonesetal.2000,
Inhibitorsofapoptosisproteins(IAPs)areafamilyof Qiuetal.2004).However,onlylimitedinformationis
potentantiapoptoticproteinsthatwereoriginallydis- available on apoptotic regulation in mosquitoes, de-
covered in insect baculoviruses (Cydia pomonella spitethecentralrolethatthesevectorsplayindisease
granulosis virus and Orgyia pseudotsugata nuclear transmission. Here, we present the developmental
polyhedrosisvirus)(Crooketal.1993,Birnbaumetal. regulation of an IAP1 homolog from Aedes aegypti
1994,ClemandMiller1994),andtheyhavesincebeen (L.),AaeIAP1,thevectorofyellowfeveranddengue
identiÞedinvariousotherviruses(Chaconetal.1995, viruses,bothofwhichcancauseseverehumanmor-
Delhonetal.2006),yeast(Walteretal.2006),inver- bidityandmortality(Patterson1992,Huhtamoetal.
tebrates(Hayetal.1995,Huangetal.2001,Muroet 2006). Furthermore, we report that the expression
al.2002),andvertebrates(Digbyetal.1996,Listonet levelofAaeIAP1transcriptisinducedtosigniÞcantly
al.1996,Ambrosinietal.1997,Vitte-Monyetal.1997, (P(cid:1)0.001)higherlevelsinAe.aegyptiunderstressful
Wagenknechtetal.1999).ManyIAPsarecapableof environmental conditions (e.g., UV radiation, heat-
blocking apoptosis when they are overexpressed in shock,andpesticidetreatment).ThisistheÞrstreport
cellsofotherspecies(Beidleretal.1995,Hawkinset ofthedevelopmentalandenvironmentalregulationof
al.1996,Hawkinsetal.1998,Lietal.2007),suggesting anIAPhomologinAe.aegypti.
that IAPs target a conserved step in the apoptosis
pathway.
MaterialsandMethods
Sampling for Different Developmental Stages of
Theuseoftrade,Þrm,orcorporationnamesinthispublicationis Ae. aegypti. The Orlando strain of Ae. aegypti was
fortheinformationandconvenienceofthereader.Suchusedoesnot rearedintheinsectaryoftheMosquitoandFlyRe-
constituteanofÞcialendorsementorapprovalbytheUnitedStates searchUnitatCenterforMedical,Agricultural,and
DepartmentofAgricultureortheAgriculturalResearchServiceof
Veterinary Entomology, ARSÐUSDA in Gainesville,
anyproductorservicetotheexclusionofothersthatmaybesuitable.
1E-mail:[email protected]. FL.Mosquitoeswererearedusingmethodsdescribed
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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Developmental and Environmental Regulation of AaeIAP1 Transcript in
5b. GRANT NUMBER
Aedes aegypti
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6. AUTHOR(S) 5d. PROJECT NUMBER
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USDA -ARS,Center for Medical, Agricultural, and Veterinary REPORT NUMBER
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1072 JOURNALOFMEDICALENTOMOLOGY Vol.45,no.6
previously(Pridgeonetal.2007).Brießy,eggswere replicatedthreetimesandsampleswerestoredat(cid:3)80(cid:2)C
hatchedinaßask,andthelarvaewereheldovernight forlaterRNAisolation.
intheßaskandthentransferredtoaplastictraycon- Ae. aegypti RNA Extraction and cDNA Synthesis.
tainingdistilledwater.Larvaldietwasaddedtoeach TotalRNAwasisolatedusingTRIzolreagent(Invitro-
tray. Mosquitoes were reared in an environmental gen,Carlsbad,CA)followingthemanufacturerÕspro-
chamber set with a temperature ranging from 26 to tocol.Brießy,0.8mlofTRIzolreagentwasaddedto
28(cid:2)Cand80%RH.Incandescentlightingwassettoa each sample (80 mg). Samples were homogenized
crepuscularproÞlewithaphotoperiodof14:10(L:D) using a Tissuemiser (Fisher), and then they were
h,including2hofsimulateddawnanddusk.Larval incubatedatroomtemperaturefor5min.Chloroform
sampleswerecollectedatdifferenttimesafterhatch- (0.2ml)wasaddedtoeachsample,andthemixture
ing.Ateachtime,sixvouchersampleswerecollected, wasvortexedfor1minandkeptatroomtemperature
witheachvouchercontaining20Ð30mosquitolarvae. for3min.Aftercentrifugationat12,000(cid:4)gfor15min
Three vouchers were quickly frozen and stored at at 4(cid:2)C, the clear supernatant (containing the RNA)
(cid:3)80(cid:2)CforlaterRNAisolation,andtheremainderof wastransferredto1.5-mlmicrocentrifugetubes.RNA
the vouchers were used to measure the transverse precipitationwascarriedoutbyadding0.5mlofiso-
diameteroftheheadcapsule,whichhasbeenareli- propanol to each sample followed by incubation at
ablemethodforidentiÞcationoflarvalinstarsaccord- room temperature for 30 min and centrifugation at
ingtoChristophers(1960).Thetransversediameterof 12,000(cid:4)gfor15min.TheRNApelletswerewashed
theheadcapsulewasmeasuredunderamicroscope with 1 ml of 75% ethanol in diethyl pyrocarbonate
(modelStemiSV8,CarlZeiss,Thornwood,NY)con- (DEPC)-treatedwater,followedbycentrifugationat
nectedtoacamera(model11.2ColorMosaic,Diag- 12,000 (cid:4) g for 10 min. The pellets were dried and
nosticInstruments,SterlingHeights,MI).Pupalsam- suspendedinanappropriatevolumeofDEPC-water
pleswerecollectedatdifferenttimestogetearlystage accordingtothesizeofthepellet((cid:5)10Ð50(cid:1)l).Total
pupae,middlestagepupae,andlatestagepupaesam- RNAwasthentreatedwithampliÞcationgradeDNa-
ples and stored at (cid:3)80(cid:2)C for later RNA isolation. seI(Invitrogen).RNAethanolprecipitationwascar-
Adultswereheldinascreenedcageandprovided10% riedoutafteradding1/10volumeof5MNaCltothe
sucroseadlibitum.Emergedadultsweretransferred DNaseIdigestedRNAsolution.TheRNApelletswere
toanewscreenedcage,andadultsampleswerecol- then washed with 1 ml of 75% ethanol in DEPC-
lected at different times after emergence. Bovine treatedwater,followedbycentrifugationat12,000(cid:4)
bloodin1%heparinplacedinapigintestinewarmed gfor10min.Thepelletsweredriedandsuspendedin
to37(cid:2)Cwasprovidedtoadultstwiceaweek.Freshly anappropriatevolumeofDEPCwateraccordingto
laid eggs were collected, and eggs were obtained at thesizeofthepellet((cid:5)10Ð50(cid:1)l).A1.0%denaturing
differenttimes(1,3,and6dold)andstoredat(cid:3)80(cid:2)C. agarose/formaldehydegelwasusedtotesttheinteg-
MosquitoTreatmentswithUVRadiation,Highor rityandqualityofthetotalRNA.TheRNAconcen-
LowTemperature,orPesticidePermethrin.ForUV trationwasassessedbytheabsorbanceofRNAat260
treatment,thirty7-d-oldfemalessortedinto4-ozplas- nminaGenesys10UVspectrophotometer(Thermo
ticcupsandcoveredbycottonmeshweresupplied ScientiÞc,Hampton,NH).ForÞrst-standcDNAsyn-
with10%sucroseandplacedonaUVtransilluminator thesis,a20-(cid:1)lreactionmixturecontainingtotalRNA
(Fisher,Hampton,NH)forUVradiationat312nm. (3(cid:1)g),oligo(dT) primer(50(cid:1)M)(Invitrogen),and
20
Sampleswerecollectedatdifferenttimes(15min,30 10mMdNTPmixwasincubatedat70(cid:2)Cfor10minand
min,and2hpostirradiation).Fortemperaturetreat- thenplacedonice.Afterbrießyspinningthereaction
ments,thirty7-d-oldfemalessortedinto4-ounceplas- mixture,5XcDNAsynthesisbuffer,0.1Mdithiothre-
ticcupsandsuppliedwith10%sucrosewereplacedin itol, RNase inhibitor (40 U/(cid:1)l), and Cloned avian
incubatorsat4,42,or55(cid:2)C.Sampleswerecollectedat myeloblastosis virus reverse transcriptase (15 U/(cid:1)l)
different times. For the treatment with permethrin, (Invitrogen)wereaddedtothereactionmixture.The
this pesticide was diluted in acetone and topically mixture was then incubated at 42(cid:2)C for 58 min fol-
appliedatasublethaldosetoindividualmosquitoes. lowed by 95(cid:2)C for 15 min in a 9600 thermocycler
Before permethrin treatment, thirty 7-d-old females (AppliedBiosystems,FosterCity,CA).
werebrießyanesthetizedfor30swithcarbondioxide Quantitative Polymerase Chain Reaction (PCR).
andplacedona4(cid:2)Cchilltable(BioQuipProducts,Ran- TreatedAe.aegyptiwerefrozenat(cid:3)80(cid:2)Cforsubse-
choDominguez,CA).Adropletof0.5(cid:1)lofpermethrin quentRNAextraction.TotalRNAwaspreparedand
solutionatsublethaldose(2.5(cid:4)10(cid:3)5(cid:1)g)wasappliedto reversetranscribedasdescribedabove.Quantitative
thedorsalthoraxofthemosquitobyusinga700series PCR was performed with SYBR Green PCR Master
syringe and a PB 600 repeating dispenser (Hamilton, MixonanABI7300quantitativePCRSystem(Applied
Reno,NV).Samplestreatedwithpermethrinwerecol- Biosystems,FosterCity,CA).Primersfortheampli-
lectedat0.5,3,6,12,and24hafterapplication.Control ÞcationoftheAaeIAP1geneweretheforwardprimer
sampleswith0.5(cid:1)lofacetonetreatmentwerealsocol- IAP-911 F (5(cid:6)-CCTCAAAGACTGGGAAGCTG-3(cid:6))
lected at the same times as those receiving the per- and the reverse primer IAP-1133R (5(cid:6)-TGACT-
methrin treatment. After treatment, mosquitoes were GAAGCGAGGATGTTG-3(cid:6)).PrimersfortheampliÞ-
kept in plastic cups and supplied with a 10% sucrose cation of the actin gene were forward primer set
solution.Temperatureandrelativehumidityweremain- Actin-152F(5(cid:6)-AGGACTCGTACGTCGGTGAC-3(cid:6))
tainedat26(cid:2)Cand80%,respectively.Alltreatmentswere andActin-590R(5(cid:6)-CGTTCAGTCAGGATCTTC-3(cid:6)).
November2008 PRIDGEONETAL.:DEVELOPMENTALEXPRESSIONOFAaeIAP1 1073
Fig.1. RepresentativepictureshowingtheheadcapsuleofinstarsIÐIV.(a)InstarIwiththetransversediameter
oftheheadcapsuleof0.25mm.(b)InstarIIwiththetransversediameteroftheheadcapsuleof0.45mm.(c)instar
IIIwiththetransversediameteroftheheadcapsuleof0.71mm.(d)InstarIVwiththetransversediameterofthehead
capsuleof1.00mm.
TherelativeexpressionlevelofAaeIAP1wasnormal- ysisofvariance(ANOVA)byusingSigmaStatstatis-
izedtoactinlevelandcalculatedusingtheequation tical analysis software (SAS Institute, Cary, NC)
100(cid:4)2(cid:3)(cid:7)CT,where(cid:7)C (cid:8)C(AaeIAP1)(cid:3)C (actin) wherethedifferencesinthemeanvalueswereana-
t t t
(Portereikoetal.2006).Datawereanalyzedbyanal- lyzedstatistically.
Table1. Transversediametersoflarvaeheadcapsules
Samplestage Sample Sample Transversediametersoftheheadcapsules(mm) Mean(cid:9)SD
name time 1 2 3 4 5 6 7 8 9 10
Firstinstar Larvae1 6hpha NDb ND ND ND ND ND ND ND ND ND ND
Larvae2 9hph ND ND ND ND ND ND ND ND ND ND ND
Larvae3 23hph 0.30 0.34 0.25 0.28 0.32 0.28 0.31 0.29 0.32 0.27 0.296(cid:9)0.027
Larvae4 30hph 0.25 0.27 0.32 0.26 0.26 0.28 0.31 0.30 0.29 0.28 0.282(cid:9)0.023
Larvae5 33hph 0.32 0.25 0.29 0.24 0.31 0.29 0.27 0.26 0.26 0.32 0.281(cid:9)0.029
Larvae6 36hph 0.29 0.31 0.30 0.29 0.28 0.32 0.24 0.22 0.23 0.27 0.275(cid:9)0.034
Larvae7 39hph 0.20 0.30 0.31 0.30 0.27 0.24 0.25 0.26 0.28 0.30 0.271(cid:9)0.035
Secondinstar Larvae8 48hph 0.49 0.51 0.49 0.45 0.52 0.43 0.46 0.42 0.49 0.47 0.476(cid:9)0.033
Larvae9 51hph 0.42 0.48 0.44 0.42 0.45 0.45 0.50 0.41 0.47 0.52 0.456(cid:9)0.036
Larvae10 54hph 0.49 0.47 0.42 0.44 0.46 0.48 0.52 0.45 0.51 0.45 0.469(cid:9)0.031
Larvae11 57hph 0.50 0.49 0.47 0.45 0.45 0.44 0.49 0.38 0.39 0.42 0.448(cid:9)0.042
Larvae12 60hph 0.42 0.44 0.44 0.42 0.50 0.43 0.49 0.46 0.48 0.45 0.453(cid:9)0.029
Larvae13 63hph 0.44 0.49 0.75 0.43 0.70 0.49 0.44 0.47 0.44 0.60 0.475(cid:9)0.056
Thirdinstar Larvae14 72hph 0.80 0.49 0.68 0.76 0.80 0.78 0.78 0.76 0.80 0.77 0.770(cid:9)0.037
Larvae15 75hph 0.70 0.47 0.46 0.74 0.75 0.71 0.72 0.77 0.74 0.71 0.730(cid:9)0.024
Larvae16 78hph 0.76 0.78 0.74 0.71 0.72 0.77 0.73 0.80 0.70 0.77 0.748(cid:9)0.033
Larvae17 81hph 0.78 0.73 0.68 0.72 0.73 0.74 0.72 0.71 0.69 0.62 0.712(cid:9)0.042
Larvae18 84hph 0.93 0.66 0.73 0.71 0.69 0.68 0.61 0.76 0.71 0.60 0.683(cid:9)0.053
Larvae19 87hph 0.74 0.69 0.71 0.77 0.73 0.87 0.93 0.67 0.72 0.96 0.737(cid:9)0.062
Fourthinstar Larvae20 99hph 0.69 0.88 1.03 0.91 0.89 1.08 1.02 0.93 0.98 0.93 0.961(cid:9)0.070
Larvae21 105hph 0.97 0.98 0.93 1.04 1.02 1.06 0.99 1.11 0.88 0.89 0.987(cid:9)0.074
Larvae22 111hph 0.96 1.01 0.94 0.94 0.90 0.97 0.81 0.97 0.93 1.06 0.949(cid:9)0.066
Larvae23 129hph 0.96 0.96 0.92 1.00 0.96 1.02 1.07 0.98 0.87 0.92 0.977(cid:9)0.048
Larvae24 132hph 0.93 0.98 0.95 0.97 0.91 0.96 0.97 0.92 0.93 0.93 0.945(cid:9)0.024
Larvae25 145hph 0.98 0.97 0.99 0.99 1.04 1.06 1.05 0.06 1.00 0.96 0.910(cid:9)0.301
Larvae26 148hph 1.01 1.08 1.09 1.01 1.07 1.03 1.00 1.12 1.11 0.99 1.051(cid:9)0.048
Numbersinitalicsarenotincludedinthestatisticanalysis.
aPosthatch.
bNotdetermined.
1074 JOURNALOFMEDICALENTOMOLOGY Vol.45,no.6
Table2. ExpressionofAaeIAP1atdifferentdevelopmentalstages
Sample Cyclethreshold(Ct)(cid:9)SD RelativeAaeIAP1expressionlevel
Samplestage Sampletime
name Actin AaeIAP1 (cid:7)Ct-1 (cid:7)Ct-2 (cid:7)Ct-3 100(cid:4)2(cid:3)(cid:7)Ct(cid:9)SD
Egg Egg1 1d 19.520(cid:9)0.073 23.705(cid:9)0.038 4.185 3.480 3.440 7.891(cid:9)2.077
Egg2 3d 19.345(cid:9)0.055 21.970(cid:9)0.001 2.625 2.970 2.440 15.801(cid:9)2.855
Egg3 6d 22.215(cid:9)0.121 24.095(cid:9)0.021 1.880 1.980 2.000 25.839(cid:9)1.164
Firstinstar Larvae1 6ha 17.415(cid:9)0.022 21.175(cid:9)0.023 3.760 3.730 3.610 7.703(cid:9)0.431
Larvae2 9h 17.405(cid:9)0.009 20.765(cid:9)0.050 3.360 3.791 3.018 9.769(cid:9)2.560
Larvae3 23h 21.250(cid:9)0.000 23.435(cid:9)0.060 2.185 2.246 2.200 21.613(cid:9)0.473
Larvae4 30h 16.825(cid:9)0.018 22.280(cid:9)0.028 5.455 6.110 5.832 1.828(cid:9)0.421
Larvae5 33h 16.245(cid:9)0.047 20.995(cid:9)0.017 4.750 5.160 5.210 3.072(cid:9)0.560
Larvae6 36h 16.065(cid:9)0.033 21.265(cid:9)0.162 5.200 5.250 5.150 2.721(cid:9)0.094
Larvae7 39h 16.895(cid:9)0.019 21.525(cid:9)0.162 4.630 4.846 4.600 3.880(cid:9)0.351
Secondinstar Larvae8 48h 19.580(cid:9)0.026 22.185(cid:9)0.006 2.605 2.449 2.629 16.975(cid:9)1.173
Larvae9 51h 16.980(cid:9)0.028 22.130(cid:9)0.022 5.150 5.454 5.190 2.621(cid:9)0.289
Larvae10 54h 18.150(cid:9)0.026 21.390(cid:9)0.014 3.240 3.071 3.300 10.879(cid:9)0.910
Larvae11 57h 21.245(cid:9)0.023 24.130(cid:9)0.039 2.885 3.030 3.130 12.401(cid:9)1.066
Larvae12 60h 17.570(cid:9)0.026 22.355(cid:9)0.063 4.785 5.150 5.050 3.154(cid:9)0.422
Larvae13 63h 17.970(cid:9)0.015 22.735(cid:9)0.049 4.765 5.830 5.430 2.585(cid:9)0.987
Thirdinstar Larvae14 72h 16.730(cid:9)0.003 22.205(cid:9)0.007 5.475 5.970 5.927 1.829(cid:9)0.364
Larvae15 75h 17.670(cid:9)0.076 22.805(cid:9)0.020 5.135 6.542 5.442 2.073(cid:9)0.908
Larvae16 78h 17.905(cid:9)0.019 22.570(cid:9)0.003 4.665 6.590 5.040 2.673(cid:9)1.486
Larvae17 81h 18.005(cid:9)0.031 22.990(cid:9)0.046 4.985 4.437 5.141 3.536(cid:9)0.949
Larvae18 84h 17.350(cid:9)0.041 22.345(cid:9)0.205 4.995 5.439 5.340 2.637(cid:9)0.440
Larvae19 87h 17.460(cid:9)0.020 23.065(cid:9)0.207 5.605 5.578 5.540 2.099(cid:9)0.047
Fourthinstar Larvae20 99h 18.235(cid:9)0.015 22.140(cid:9)0.045 3.905 3.904 4.145 6.336(cid:9)0.592
Larvae21 105h 19.125(cid:9)0.040 22.510(cid:9)0.179 3.385 3.081 3.140 10.912(cid:9)1.185
Larvae22 111h 19.110(cid:9)0.073 22.110(cid:9)0.030 3.000 2.775 2.720 14.096(cid:9)1.411
Larvae23 129h 20.380(cid:9)0.041 23.000(cid:9)0.018 2.620 2.775 2.652 15.598(cid:9)0.873
Larvae24 132h 19.430(cid:9)0.040 22.845(cid:9)0.059 3.415 3.440 3.650 8.852(cid:9)0.771
Larvae25 145h 19.540(cid:9)0.003 22.885(cid:9)0.389 3.345 3.975 3.726 7.919(cid:9)1.769
Larvae26 148h 19.855(cid:9)0.027 22.815(cid:9)0.003 2.960 2.602 2.544 15.491(cid:9)2.311
Pupa P1 154h 18.230(cid:9)0.028 20.125(cid:9)0.004 1.895 1.825 1.623 29.196(cid:9)2.916
P2 157h 19.100(cid:9)0.031 20.565(cid:9)0.086 1.465 1.600 1.273 36.860(cid:9)4.226
P3 169h 19.105(cid:9)0.035 20.440(cid:9)0.039 1.335 1.473 1.090 40.881(cid:9)5.586
Adult A1 1d 19.950(cid:9)0.294 20.290(cid:9)0.014 0.340 0.540 0.140 79.511(cid:9)10.996
A2 6d 19.015(cid:9)0.004 20.705(cid:9)0.120 1.690 1.735 1.780 30.051(cid:9)0.938
A3 12d 18.605(cid:9)0.007 20.005(cid:9)0.049 1.400 1.170 1.370 40.341(cid:9)3.574
aHoursposthatch.
Results To understand the developmental regulation of
AaeIAP1transcriptunderdifferentphysiologicalcon-
To identify the instars of the larval samples, the
ditions, quantitative PCR was performed using egg,
transversediametersoftheheadcapsulesweremea-
larval,pupal,andadultsamples.AsshowninTable2
sured(Fig.1).AccordingtoChristophers(1960),the
andFig.2,theexpressionofAaeIAP1transcriptwas
mostdependablemethodforidentiÞcationoflarvaeis
detectable in all four developmental stages. Our re-
by measuring the transverse diameter of the head
sultsrevealedthat,althoughtheexpressionofAaeIAP1
capsule.ThesuccessivelarvalinstarsIÐIVofthehead
diameter should be (cid:5)0.3, 0.45, 0.65, and 0.95 mm, transcript was detectable in all life stages of Ae. ae-
respectively.Usingthecriterionof(cid:5)0.3mmforinstar gypti, the expression levels of AaeIAP1 at each indi-
vidual life stage were different. For example, in the
I,seventimepointsamples(L1ÐL7)upto39hpost-
hatchwereidentiÞedtobeÞrstinstar,withmeanhead embryonicstage,AaeIAP1wasexpressedsigniÞcantly
capsuletransversediameterrangingfrom0.27to0.296 (P(cid:1)0.001)higherinthelatterperiod(6dold)than
mm(Table1).Similarly,usingthecriterionof(cid:5)0.45 intheearlierperiod(1dold)orthemiddle(3dold)
mm for instar II, six time points (L8ÐL13) ranging (Table2;Fig.2).AlthoughAaeIAP1wasdetectablein
from48to63hposthatchwereidentiÞedassecond alllarvalstages,itsexpressionlevelinthefourthinstar
instars,withmeanheadcapsuletransversediameters wassigniÞcantly(P(cid:1)0.001)higherthanthatinthe
rangingfrom0.448to0.476mm(Table1).Usingthe thirdinstar.Ourresultsalsorevealedthattheexpres-
criterion of (cid:5)0.65 mm for instar III, six time point sion level of AaeIAP1 transcript in pupal and adult
samples(L14ÐL19)rangingfrom72to87hposthatch stagesweresigniÞcantly(P(cid:1)0.001)higherthanthat
wereidentiÞedasthirdinstars,withmeanheadcap- inthelarvalinstars(Table2;Fig.2).
suletransversediametersrangingfrom0.683to0.770 Tounderstandwhetherextremeloworhightem-
mm (Table 1). Using the criterion of (cid:5)0.95 mm for peratures affect the expression of AaeIAP1, we per-
instarIV,seventimepointsamples(L20ÐL26)ranging formedquantitativePCRtocomparetheexpression
from99to148hposthatchwereidentiÞedasfourth levels of AaeIAP1 transcript in different samples. As
instars,withmeanheadcapsuletransversediameters showninTable3andFig.3,theexpressionlevelsof
rangingfrom0.945to1.051mm(Table1). AaeIAP1transcriptwereincreasedsigniÞcantly(P(cid:1)
November2008 PRIDGEONETAL.:DEVELOPMENTALEXPRESSIONOFAaeIAP1 1075
Fig.2. RelativeexpressionlevelsofAaeIAP1transcriptinegg,larval,pupal,andadultstagesdetectedbyquantitativePCR.
TheagesofinstarI(sample1Ð7)are6,9,23,30,33,36,and39hposthatch,respectively.TheagesofinstarII(sample8Ð13)
are48,51,54,57,60,and63hposthatch,respectively.TheagesofinstarIII(sample14Ð19)are72,75,78,81,84,and87h
posthatch,respectively.TheagesofinstarIV(sample20Ð26)are99,105,111,129,132,145,and148hposthatch,respectively.
Threesamplesofegg,pupal,andadultstagesarerepresentativesamplesofearly,middle,andlatestages.
0.001) upon the treatment of low (4(cid:2)C) and high To understand whether UV light exposure will
(42(cid:2)C)temperaturesat3hposttreatment.Whenwe affect the expression of AaeIAP1, we performed
set the AaeIAP1 level in the untreated mosquito as quantitativePCRafterexposingadultmosquitoesto
standard (cid:8) 1, the treatment of low (4(cid:2)C) and high UVradiation.OurresultsrevealedthatUVradiation
(42(cid:2)C)temperaturesfor3hincreasedtheexpression alsosigniÞcantly(P(cid:1)0.001)inducedtheexpression
of AaeIAP1 by (cid:5)two-fold. The highest increase of levelofAaeIAP1transcript(Table3;Fig.4).Thirty
AaeIAP1expressionlevelwasobservedinmosquitoes minutesafterexposuretoUVradiation,theexpres-
treatedatatemperatureof55(cid:2)Cfor20min(Table3; sion level of AaeIAP1 transcript was increased ap-
Fig. 3), which increased the expression level of proximatelyeight-foldcomparedwiththeuntreated
AaeIAP1transcriptby(cid:5)18-fold. control.
Table3. ExpressionofAaeIAP1underdifferentstressconditions
Cyclethreshold(Ct)(cid:9)SD RelativeAaeIAP1expressionlevel
Samplename
Actin AaeIAP1 (cid:7)Ct-1 (cid:7)Ct-2 (cid:7)Ct-3 100(cid:4)2(cid:3)(cid:7)Ct(cid:9)SD
Untreated 19.065(cid:9)0.018 21.050(cid:9)0.026 1.985 2.020 1.300 30.177(cid:9)9.043
4(cid:2)C-0.5h 19.040(cid:9)0.395 19.505(cid:9)0.465 0.465 1.040 1.340 53.527(cid:9)17.009
4(cid:2)C-3h 20.695(cid:9)0.063 20.900(cid:9)0.563 0.205 0.830 0.560 70.279(cid:9)15.397
4(cid:2)C-6h 18.130(cid:9)0.009 19.885(cid:9)0.052 1.755 1.590 1.620 31.793(cid:9)1.906
4(cid:2)C-12h 16.980(cid:9)0.012 19.375(cid:9)0.120 2.395 2.470 2.350 18.892(cid:9)0.790
4(cid:2)C-24h 17.965(cid:9)0.063 20.535(cid:9)0.009 2.530 2.120 2.220 20.594(cid:9)2.943
42(cid:2)C-0.5h 17.720(cid:9)0.011 20.315(cid:9)0.067 2.650 2.340 2.540 17.626(cid:9)1.946
42(cid:2)C-3h 20.705(cid:9)0.118 21.745(cid:9)0.080 1.180 0.900 0.140 62.825(cid:9)24.643
42(cid:2)C-6h 17.855(cid:9)0.064 19.960(cid:9)0.019 2.105 2.020 2.070 23.906(cid:9)0.710
42(cid:2)C-12h 18.695(cid:9)0.051 20.600(cid:9)0.043 1.905 1.980 1.960 25.918(cid:9)0.701
42(cid:2)C-24h 30.905(cid:9)0.101 31.735(cid:9)0.931 0.830 0.230 0.550 69.939(cid:9)14.574
55(cid:2)C-20min 36.220(cid:9)0.058 33.820(cid:9)0.329 (cid:3)2.40 (cid:3)2.43 (cid:3)2.45 535.197(cid:9)6.403
UV-15min 26.920(cid:9)0.137 27.695(cid:9)0.024 0.775 0.660 0.860 58.941(cid:9)4.119
UV-30min 35.655(cid:9)0.135 34.440(cid:9)0.009 (cid:3)1.215 (cid:3)1.130 (cid:3)1.430 240.149(cid:9)26.227
UV-120min 28.370(cid:9)0.510 27.040(cid:9)0.007 (cid:3)1.330 (cid:3)0.980 (cid:3)1.280 230.496(cid:9)29.112
Acetone-0.5h 17.925(cid:9)0.022 19.960(cid:9)0.126 2.035 2.020 1.930 25.100(cid:9)0.998
Acetone-3h 20.580(cid:9)0.003 22.420(cid:9)0.024 1.840 2.210 2.220 23.670(cid:9)3.692
Acetone-6h 21.215(cid:9)0.008 22.250(cid:9)0.037 1.035 1.190 1.070 46.755(cid:9)2.599
Acetone-12h 27.290(cid:9)0.228 28.005(cid:9)0.006 0.715 1.020 0.970 53.761(cid:9)6.261
Acetone-24h 19.480(cid:9)0.071 21.130(cid:9)0.056 1.650 1.750 1.740 30.510(cid:9)1.177
Permethrin-0.5h 25.085(cid:9)0.009 26.370(cid:9)0.083 1.285 1.310 1.230 41.334(cid:9)1.178
Permethrin-3h 33.025(cid:9)0.578 32.170(cid:9)0.590 (cid:3)0.855 (cid:3)0.810 (cid:3)1.340 203.116(cid:9)43.421
Permethrin-6h 33.280(cid:9)0.099 27.830(cid:9)0.022 (cid:3)5.45 (cid:3)5.86 (cid:3)5.21 5860.752(cid:9)1516
Permethrin-12h 18.860(cid:9)0.011 20.355(cid:9)0.388 1.495 1.030 1.760 37.991(cid:9)9.964
Permethrin-24h 26.535(cid:9)0.011 27.400(cid:9)0.196 0.865 0.720 0.940 55.913(cid:9)4.381
1076 JOURNALOFMEDICALENTOMOLOGY Vol.45,no.6
Fig.3. RelativeexpressionlevelsofAaeIAP1transcriptin
Fig.5. RelativeexpressionlevelsofAaeIAP1transcriptin
responsetodifferenttemperaturetreatments.Datarepre- responsetoacetonetreatment.Datarepresentmean(cid:9)SD
sent mean (cid:9) SD from three replicates. The asterisk (*)
fromthreereplicates.Theasterisk(*)indicatestherewasa
indicates there was a signiÞcant difference between the
signiÞcant difference between the treatment and the un-
treatmentandtheuntreatedcontrol.
treatedcontrol.
Next,weexaminedtheeffectofacetonetreatment
creasedsigniÞcantly(P(cid:1)0.05).Thehighestincrease
ontheexpressionofAaeIAP1.AsshowninTable3and
ofAaeIAP1expressionlevelwasobservedat6hpost-
Fig.5,acetonetreatmentdidnotaffecttheexpression
permethrintreatment((cid:5)200-foldgreaterthaninthe
ofAaeIAP1at0.5and3hposttreatment.However,at
6and12hposttreatment,acetonesigniÞcantly(P(cid:1) untreatedcontrol)(Table3;Fig.6).
0.001)increasedtheexpressionofAaeIAP1,within-
creased level of AaeIAP1 by approximately two-fold Discussion
(Table3;Fig.5).At24hposttreatmentwithacetone,
Toidentifytheinstars,thetransversediametersof
theexpressionlevelsofAaeIAP1werenotsigniÞcantly
(P(cid:10)0.05)differentbetweentheuntreatedandthe theheadcapsuleweremeasured.AccordingtoChris-
tophers(1960),thesuccessiveinstarsIÐIVofthehead
acetone-treatedsamples.
diameter should be (cid:5)0.3, 0.45, 0.65, and 0.95 mm,
Todeterminetheeffectofpermethrinontheex-
respectively.Ourresultsrevealedthatthemeanhead
pressionlevelofAaeIAP1transcript,wetopicallyap-
diametersofinstarsIÐIVwere(cid:5)0.28,0.46,0.72,and
pliedpermethrintoadultmosquitoesandperformed
0.98mm,respectively,whichwasonlyslightlydiffer-
quantitativePCR.Ourresultsrevealedthat,at0.5h
entfromthecriteriasetbyChristophers(1960).We
postpermethrintreatment,AaeIAP1expressionlevels
were not signiÞcantly different (P (cid:10) 0.05) between alsonoticedadifferenceinthedurationofeachinstar.
For example, our Þrst instar lasted up to 39 h post-
untreatedandpermethrin-treatedmosquitoes(Table
hatch,whereastheÞrstinstarofAe.aegypidescribed
3;Fig.6).However,at3hpostpermethrintreatment,
by Christophers (1960) lasted 28 h. The difference
the expression levels of AaeIAP1 transcript were in-
couldbeduetodifferentrearingconditions,suchas
thewatertemperatureandavailabilityoffood.
Inthecurrentstudy,wedemonstratedtheexpres-
sionofAaeIAP1indifferentdevelopmentalstagesof
Ae.aegyptibyquantitativePCR.Ourresultsrevealed
thatAaeIAP1wasexpressedthroughoutthedevelop-
mentalstagesofAe.aegypti,whichisconsistentwith
previousreportonanIAP1homologexpressedinall
life stages of Aedes triseriatus (Say) and Ae. aegypti
(Blitvichetal.2002,Becketal.2007).However,the
expression level of AaeIAP1 in late embryonic stage
was signiÞcantly higher than that in the early and
middleembryonicstages.Thisobservationisofgreat
importanceinthatitsuggeststhatAaeIAP1mightplay
a functional role in embryonic development in Ae.
aegypti.OurresultsalsorevealedthatAaeIAP1expres-
sionlevelsinpupalandadultstagesweresigniÞcantly
Fig.4. RelativeexpressionlevelsofAaeIAP1transcriptin
responsetoUVirradiation.Datarepresentmean(cid:9)SDfrom higherthanthatinthelarvalstage.Furthermore,at
threereplicates.Theasterisk(*)indicatestherewasasig- differenttimeswithineachdevelopmentalstage,the
niÞcantdifferencebetweenthetreatmentandtheuntreated expressionlevelsofAaeIAP1weredifferent,suggest-
control. ingthatAaeIAP1playsapivotalrolethroughoutthe
November2008 PRIDGEONETAL.:DEVELOPMENTALEXPRESSIONOFAaeIAP1 1077
Fig.6. RelativeexpressionlevelsofAaeIAP1transcriptinresponsetopermethrintreatment.Datarepresentmean(cid:9)SD
fromthreereplicates.Theasterisk(*)indicatestherewasasigniÞcantdifferencebetweenthetreatmentandtheuntreated
control.
physiological process of the development of Ae. Several IAP genes have been demonstrated to be
aegypti. capableofpreventingprogrammedcelldeathinduced
Thetranscriptionalregulationofgeneexpressionis byUVirradiation.Forexample,whentheIAPgene
aprimarymeansbywhichinsectsadapttoachanging fromOrgyiapseudotsugatanuclearpolyhedrosisvirus
environment (Liu et al. 2007). Gene regulation in was overexpressed in insect cells, the UV radiation-
response to extreme temperatures has been exten- induced apoptosis was blocked (Manji et al. 1997).
sivelystudiedininsects.Forexample,inresponseto However,informationonIAPgeneregulationinor-
coldshockandheatshock,a23-kDasmallheatshock ganismsafterUVirradiationisunknown.Ourstudy,
proteintranscriptwasexpressedmuchhigherinnon- for the Þrst time, demonstrated that UV irradiation
diapausingßeshßySarcophagacrassipalpisMacquart signiÞcantly increased the expression of AaeIAP1 in
(Yocumetal.1998).However,generegulationstudies Ae. aegypti. This result is consistent with previous
inresponsetocoldorheatshockhavebeenmainly humancancercellstudiesonexpressionlevelofsur-
focusedonheatshockproteintranscript.Knowledge vivin (an IAP homolog in human), which demon-
ofresponseofothergenestocoldandheatshockis strated that survivin expression level is signiÞcantly
verylimited.Inthisstudy,wedemonstratedthatAae- higherinUV-inducedmelanomacellsthaninnormal
IAP1 gene expression was induced to signiÞcantly cells(Rajetal.2008).Theup-regulationofAaeIAP1
higherlevelsinresponsetobothcoldandheatshock, followedbyUVradiationstronglysuggeststhatAae-
suggestingthatAaeIAP1mightplayanimportantrole IAP1 play a functional role in UV radiation-induced
instress-inducedapoptosis. apoptosis.
1078 JOURNALOFMEDICALENTOMOLOGY Vol.45,no.6
The primary approach used for mosquito control expressionofaninhibitorofapoptosisprotein1homo-
has mainly relied on pesticides. Because of its low loguefromAedestriseriatusmosquitoes.InsectMol.Biol.
mammalian toxicity, pyrethroid insecticides are 11:431Ð442.
widelyusedtoimpregnatebednetsandindoorresid- Chacon,M.R.,F.Almazan,M.L.Nogal,E.Vinuela,andJ.F.
ualsprayprogramsinanefforttocontrolmosquitoes. Rodriguez. 1995. TheAfricanswinefevervirusIAPho-
However,frequentuseofpyrethroidshasresultedin mologisalatestructuralpolypeptide.Virology214:670Ð
674.
developmentofresistanceinÞeldpopulations(Jinfu
Christophers,S.R. 1960. Aedesaegypti(L.)theyellowfever
1999, Wang 1999). Gene regulation has been exten-
mosquito:itslifehistory,bionomicsandstructure.Cam-
sively studied in pesticide resistant mosquitoes. For
bridgeUniversityPress,Cambridge,UnitedKingdom.
example,inaninsecticideresistantstrainofAnopheles
Clem,R.J.,andL.K.Miller. 1994. Controlofprogrammed
stephensi Liston glutathione S-transferase transcripts celldeathbythebaculovirusgenesp35andiap.Mol.Cell.
andcytochromeP450geneexpressionarereportedto Biol.14:5212Ð5222.
be up-regulated (Vontas et al. 2007). However, IAP Crook,N.E.,R.J.Clem,andL.K.Miller. 1993. Anapop-
gene regulation upon pesticide treatment has never tosis-inhibitingbaculovirusgenewithazincÞnger-like
been studied. Our study, for the Þrst time, demon- motif.J.Virol.67:2168Ð2174.
stratedthatAaeIAP1geneexpressionwassigniÞcantly Delhon,G.,E.R.Tulman,C.L.Afonso,Z.Lu,J.J.Becnel,
increasedinresponsetopermethrintreatment,sug- B.A.Moser,G.F.Kutish,andD.L.Rock. 2006. Genome
gestingthatAaeIAP1mightplayapivotalroleinstress- ofinvertebrateiridescentvirustype3(mosquitoirides-
centvirus).J.Virol.80:8439Ð8449.
inducedapoptosis.
Digby,M.R.,W.G.Kimpton,J.J.York,T.E.Connick,and
Inconclusion,theexpressionofAaeIAP1transcript
J.W.Lowenthal. 1996. ITA,avertebratehomologueof
wastightlyregulatedduringthephysiologicaldevel-
IAPthatisexpressedinTlymphocytes.DNACellBiol.15:
opmentinAe.aegypti,withsigniÞcantlyhigherlevels
981Ð988.
inthepupalandadultstagescomparedwiththelarval Hawkins,C.J.,A.G.Uren,G.Hacker,R.L.Medcalf,and
stage. Furthermore, AaeIAP1 expression levels were D.L.Vaux. 1996. Inhibitionofinterleukin1beta-con-
increasedsigniÞcantlyinresponsetodifferentenvi- vertingenzyme-mediatedapoptosisofmammaliancells
ronmentalstresses(e.g.,lowandhightemperatures, by baculovirus IAP. Proc. Natl. Acad. Sci. U.S.A. 93:
UVirradiation,andpermethrintreatment),suggesting 13786Ð13790.
thatAaeIAP1notonlyplaysanimportantroleinthe Hawkins,C.J.,P.G.Ekert,A.G.Uren,S.P.Holmgreen,and
physiological process of the development of Ae. ae- D.L.Vaux. 1998. Anti-apoptoticpotentialofinsectcel-
gypti but also plays a pivotal role in stress-induced lularandviralIAPsinmammaliancells.CellDeathDiffer.
5:569Ð576.
apoptosis.Thisstudymayprovideinformationuseful
Hay,B.A.,D.A.Wassarman,andG.M.Rubin. 1995. Dro-
fordesigningnovelcontrolstrategiesformosquitoes.
sophila homologs of baculovirus inhibitor of apoptosis
proteinsfunctiontoblockcelldeath.Cell83:1253Ð1262.
Huang,Q.,Q.L.Deveraux,S.Maeda,H.R.Stennicke,B.D.
Acknowledgment
Hammock, and J. C. Reed. 2001. Cloning and charac-
WethankLynnJefferson,NeilSanscrainte,HeatherFur- terizationofaninhibitorofapoptosisprotein(IAP)from
long, and Mathew H. Brown (USDAÐARS) for technical Bombyxmori.Biochim.Biophys.Acta1499:191Ð198.
support.WealsothankDrs.S.M.VallesandM.Choi(USDAÐ Huhtamo,E.,S.Vuorinen,N.Y.Uzcategui,O.Vapalahti,H.
ARS)forcriticalreviewsofthemanuscript.Thisstudywas Haapasalo, and J. Lumio. 2006. Fatal dengue virus in-
supportedbyagrantfromtheDeployedWar-FighterPro- fectioninaFinnishtraveler.J.Clin.Virol.37:323Ð326.
tectionResearchProgramfundedbytheU.S.Departmentof Jinfu,W. 1999. ResistancetodeltamethrininCulexpipiens
DefensethroughtheArmedForcesPestManagementBoard. pallens (Diptera: Culicidae) from Zhejiang, China.
J.Med.Entomol.36:389Ð393.
Jones,G.,D.Jones,L.Zhou,H.Steller,andY.Chu. 2000.
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9:345Ð359. Received18January2008;accepted24February2008.